By the end of 2007, about 33.2 million people in the world were living infected with the human immunodeficiency virus (HIV) and more than 25 million people have died of acquired immunodeficiency syndrome (AIDS). Therefore, it is urgently needed to discover and develop new therapeutic strategies against HIV infection. So far, 28 anti-HIV drugs have been approved by the United States Food and Drug Administration to treat people infected with HIV, including 15 reverse transcriptase inhibitors (RTIs), 10 protease inhibitors (PIs), one integrase inhibitor (II), and two entry inhibitors (EIs). All the RTIs, PIs, and II inhibit HIV replication after the virus gets into the host cells, while the two EIs can block HIV entry into the host cell.
HIV entry is initiated by binding of the envelope glycoprotein (Env) surface subunit gp120 to the primary receptor CD4 and then to a chemokine receptor (CCR5 or CXCR4) on the target cell. These interactions trigger gp41 structural rearrangement, resulting in the formation of a stable gp41 six-helix bundle (6-HB) core structure, which brings the both the viral and target cell membranes into proximity for fusion. In the 6-HB, three N-terminal heptad repeats (NHR or HR1) associate to form the central trimeric coiled coil, while three C-terminal heptad repeat (CHR or HR2) pack obliquely in an anti-parallel manner into the highly conserved hydrophobic grooves on the surface of the NHR-trimer. In each groove, there is a highly conserved hydrophobic deep pocket formed by the pocket-forming sequence (residues 565-581) in the NHR region. This pocket plays a critical role in viral fusion and maintaining the stability of the 6-HB.
One of the FDA-approved EIs is a synthetic peptide designed based on the HIV-1 gp41 CHR sequence (aa 638-673), named T20 (generic name: enfuvirtide, brand name: Fuzeon® [Trimeris]). T20 contains an HR (heptad repeat)-binding domain (HBD) and a tryptophan-rich domain (TRD) (FIG. 1), through which T20 can bind to the HR-sequence, especially the GIV motif in NHR, and the target cell membrane, respectively, to inhibit HIV fusion with and entry into the target cell.
The clinical of application of T20 is limited because of the rapid emergence of T20-resistant viruses in T20-treated patients. Both in vitro and in vivo studies have shown that T20 resistance is associated with single or double mutations in the GIV and the adjacent region (aa 36-45) in the gp41 NHR domain (e.g., G36D, I37V, V38A, V38E, V38M, N42D, N42S, and N43D) because this region is the primary binding site in gp41 and these mutations impact the binding of T20 to the viral gp41 NHR region. Since the binding of T20 to the HR sequence in the NHR domain is not strong enough to compete with the interaction between the viral gp41 CHR and NHR regions, T20 has to use its C-terminal TRD to interact with the target cell membrane in order to stabilize its interaction with the viral gp41 NHR region. Another weakness of the T20 peptide as an anti-HIV drug is that it has to be administrated by injection twice daily at high dosage (90 mg/dose), resulting in painful injection-site reactions in most patients. Furthermore, because of the high production cost of peptide synthesis, T20 is exorbitantly expensive for use, especially in developing countries.
C38 is a 38-mer peptide derived from aa 626-673 of the HIV-1 gp41 CHR region. It contains a pocket-binding domain (PBD) and an HBD (FIG. 1), through which C38 binds to the pocket-forming sequence and the HR-sequence in the viral gp41 NHR region to form stable heterologous 6-HB and block the fusion-active gp41 core formation. This results in inhibition of HIV fusion with, and entry into, the host cell. Because the primary binding site of C38 is the pocket-forming sequence, rather than the aa 36-45 region, the mutations in the GIV motif and the adjacent region in the gp41 NHR domain T1144 do not significantly affect the binding of C38. Therefore, the viruses with mutations in the aa 36-45 are resistant to T20, but sensitive to C38.
T1144 is also a 38-mer peptide containing a PBD and an HBD (FIG. 1). It was designed by modifying the amino acid sequence of C38 to increase α-helicity and 6-HB stability and to improve pharmacokinetic properties. Like C38, T1144 is much more effective than T20 against both the R5 and X4 strains of HIV-1, including those resistant to T20.
One recent study has shown that the combination of a CHR peptide containing the PBDn (e.g., C34, C38, T1144) and a CHR peptide lacking the PBD (e.g., T20) exhibits potent synergistic effect against both T20-sensitive and resistant viruses.